More than 150 recombinantly produced proteins and peptides have been approved by the U.S. Food and Drug Administration (FDA) for use as biotechnology drugs and vaccines, with another 370 in clinical trials. Unlike small molecule therapeutics that are produced through chemical synthesis, proteins and peptides are most efficiently produced in living cells. However, current methods of production of recombinant proteins in bacteria often produce improperly folded, aggregated or inactive proteins, and many types of proteins require secondary modifications that are inefficiently achieved using known methods.
One primary problem with known methods lies in the formation of inclusion bodies made of aggregated proteins in the cytoplasm, which occur when an excess amount of protein accumulates in the cell. Another problem in recombinant protein production is establishing the proper secondary and tertiary conformation for the expressed proteins. One barrier is that bacterial cytoplasm actively resists disulfide bonds formation, which often underlies proper protein folding (Derman et al. (1993) Science 262:1744-7). As a result, many recombinant proteins, particularly those of eukaryotic origin, are improperly folded and inactive when produced in bacteria.
Numerous attempts have been developed to increase production of properly folded proteins in recombinant systems. For example, investigators have changed fermentation conditions (Schein (1989) Bio/Technology, 7:1141-1149), varied promoter strength, or used overexpressed chaperone proteins (Hockney (1994) Trends Biotechnol. 12:456-463), which can help prevent the formation of inclusion bodies.
Secretion
An alternative approach to increase the harvest of properly folded proteins is to secrete the protein from the intracellular environment. In Gram-negative bacteria, a protein secreted from the cytoplasm can end up in the periplasmic space, attached to the outer membrane, or in the extracellular broth. Inclusion bodies, made of aggregated proteins are usually not formed if proteins are secreted out of the cytoplasm of the cell. Secretion into the periplasmic space also has the well known effect of facilitating proper disulfide bond formation (Bardwell et al. (1994) Phosphate Microorg. 270-5; Manoil (2000) Methods in Enz. 326: 35-47). Secretion of recombinant protein is appealing because it can result in more efficient isolation of the protein; can promote proper folding and disulfide bond formation of the transgenic protein, leading to an increase in the percentage of the protein in active form; can result in reduced formation of inclusion bodies and reduced toxicity to the host cell; and can provide an increased percentage of the recombinant protein in soluble form. The potential for excretion of the protein of interest into the culture medium can also potentially promote continuous, rather than batch culture for protein production.
Gram-negative bacteria have evolved numerous systems for the active export of proteins across their dual membranes (see FIG. 1). These routes of secretion include, e.g.: the ABC (Type I) pathway, the Path/Fla (Type III) pathway, and the Path/Vir (Type IV) pathway for one-step translocation across both the plasma and outer membrane; the Sec (Type II), Tat, MscL, and Holins pathways for translocation across the plasma membrane; and the Sec-plus-fimbrial usher porin (FUP), Sec-plus-autotransporter (AT), Sec-plus-two partner secretion (TPS), Sec-plus-main terminal branch (MTB), and Tat-plus-MTB pathways for two-step translocation across the plasma and outer membranes. Not all bacteria have all of these secretion pathways.
Three protein systems (types I, III and IV) secrete proteins across both membranes in a single energy-coupled step. Four systems (Sec, Tat, MscL and Holins) secrete only across the inner membrane, and four other systems (MTB, FUP, AT and TPS) secrete only across the outer membrane.
The most common form of secretion of peptides with a signal sequence involves the Sec system. The Sec system is responsible for export of proteins with the N-terminal signal peptides across the cytoplasmic membranes (see Agarraberes and Dice (2001) Biochim Biophys Acta. 1513:1-24; Müller et al. (2001) Prog Nucleic Acid Res Mol Biol. 66:107-157). Protein complexes of the Sec family are found universally in prokaryotes and eukaryotes. The bacterial Sec system consists of transport proteins, a chaperone protein (SecB) or signal recognition particle (SRP) and signal peptidases (SPase I and SPase II). The Sec transport complex in E. coli consists of three integral inner membrane proteins, SecY, SecE and SecG, and the cytoplasmic ATPase, SecA. SecA recruits SecY/E/G complexes to form the active translocation channel. The chaperone protein SecB binds to the nascent polypeptide chain to prevent it from folding and targets it to SecA. The linear polypeptide chain is subsequently transported through the SecYEG channel and, following cleavage of the signal peptide, the protein is folded in the periplasm. Three auxiliary proteins (SecD, SecF and YajC) form a complex that is not essential for secretion but stimulates secretion up to ten-fold under many conditions, particularly at low temperatures.
Proteins that are transported into the periplasm, i.e. through a type II secretion system, can also be exported into the extracellular media in a further step. The mechanisms are generally through an autotransporter, a two partner secretion system, a main terminal branch system or a fimbrial usher porin.
Of the twelve known secretion systems in Gram-negative bacteria, eight are known to utilize targeting signal peptides found as part of the expressed protein. These signal peptides interact with the proteins of the secretion systems so that the cell properly directs the protein to its appropriate destination. Five of these eight signal-peptide-based secretion systems are those that involve the Sec-system. These five are referred to as involved in Sec-dependent cytoplasmic membrane translocation and their signal peptides operative therein can be referred to as Sec dependent secretion signals. One of the issues in developing an appropriate secretion signal is to ensure that the signal is appropriately expressed and cleaved from the expressed protein.
A signature of Sec-dependent protein export is the presence of a short (about 30 amino acids), mainly hydrophobic amino-terminal signal sequence in the exported protein. The signal sequence aids protein export and is cleaved off by a periplasmic signal peptidase when the exported protein reaches the periplasm. A typical N-terminal Sec-signal peptide contains an N-domain with at least one arginine or lysine residue, followed by a domain that contains a stretch of hydrophobic residues, and a C-domain containing the cleavage site for signal peptidases.
Bacterial protein production systems have been developed in which transgenic protein constructs are engineered as fusion proteins containing both a protein of interest and a secretion signal in an attempt to target the protein out of the cytoplasm.
Strategies have been developed to excrete proteins from the cell into the supernatant. For example, U.S. Pat. No. 5,348,867 by Georgiou focuses on expression of peptides on the surface of the cell. U.S. Pat. No. 6,329,172 to the Korea Advanced Institute of Science and Technology describes an ABC transporter in Pseudomonas fluorescens and a method of excreting proteins extracellularly through the use of this transporter co-expressed with a protein of interest. PCT Publication No. WO 96/17943 to Novo Nordisk focuses on extracellular expression of proteins from the cell by leakage from the periplasm, using portions of the sequence of A. lyticus protease I or certain Bacillus proteases linked to proteins of interest to target them to the periplasm. PCT Publication No. WO 02/40696 and U.S. Application Publication 2003/0013150, to Boehringer Ingelheim, Int., describe the extracellular expression of proteins using the bacterial signal peptide OmpA. These publications teach that OmpA interacts with SecE alone or in combination with proteins or peptides including the amino acid sequence SEGN.
Other strategies for increased expression are directed to targeting the protein to the periplasm. Some investigations focus on non-Sec type secretion (see for e.g. PCT Publication No. WO 03/079007 to the Trustees of the University of Pennsylvania; U.S. Publication No. 2003/0180937 Georgiou, U.S. Publication No. 2003/0064435 to Weiner and Turner; and PCT Publication No. WO 00/59537 to the Research Foundation of the State University of New York). However, the majority of research has focused on the secretion of exogenous proteins with a Sec-type secretion system.
A number of secretion signals have been described for use in expressing recombinant peptides or proteins. For example, U.S. Pat. No. 5,914,254 to Celtrix Pharmaceuticals, Inc. describes increased solubility and activity of proteins using fusion partners and leader sequences that are derived from interleukin-1-like proteins.
U.S. Pat. No. 4,963,495 to Genentech describes the expression of eukaryotic proteins, particularly human growth hormone, with prokaryotic signal sequences like the sequence for E. coli enterotoxin. European Patent No. 0 177 343 to Genentech describes the expression of periplasmic HGH, which can be accomplished through the use of a P. aeruginosa enterotoxin A signal sequence.
U.S. Pat. No. 5,082,783 to Biogen describes the expression of a protein such as somatomedin C, tissue plasminogen activator or tumor necrosis factor from a promoter of at most intermediate strength, such as an actin or iso-1-cytochrome c promoter operatively linked to a DNA signal sequence, such as the Mfα1 signal sequence from yeast.
PCT Publication No. WO 89/10971 by Pastan, et al., describes the expression of Pseudomonas exotoxin in E. coli using an OmpA signal sequence, and shows the differential expression of the portions of the protein in the periplasm and the medium.
U.S. Pat. No. 6,156,552 to Novo Nordisk and describes the expression in E. coli or a lipase deficient P. mendocina of a modified Pseudomonas lipase using a signal sequence. The signal sequence can be an E. coli phoA signal sequence. U.S. Pat. Nos. 6,495,357; 6,509,181; 6,524,827; 6,528,298; 6,558,939; 6,608,018; and 6,617,143 to Novozyme also describe the production of lipases, cellulases, amylases, and other enzymes, including enzymes of Pseudomonad origin, expressed with signal sequences that are preferably native to a the expressed enzyme but can also be, for example, from a Rhizomucor species, the gene for the α-factor from Saccharomyces cerevisiae or an amylase or a protease gene from a Bacillus species.
U.S. Pat. Nos. 5,595,898; 5,698,435; and 6,204,023 to Xoma describe the production of chimeric antibody fragments using a pectate lyase signal peptide. The patents disclose that pectate lyase enzymes are known in various organisms, including P. fluorescens, however no exemplification of these sequences is provided.
U.S. Pat. No. 6,258,560 to Genentech describes the expression of a DNA-digesting protein in E. coli with a bacterial signal sequence which is described as preferably native to the protein but can also be selected from the group consisting of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin leader sequences. The patent also states that the protein can also be expressed in Pseuomonads, however no exemplification is given.
PCT Publication Nos. WO 01/21662, WO 02/068660 and U.S. Application Publication 2003/0044906 to Habermann and Ertl and Aventis describe secretion of hirudin derivatives from E. coli. One of the described signal sequences is the sequence from the OprF protein of Pseudomonas fluorescens. The application identified in the '662 publication is the sequence NTLGLAIGSLIAATSFGVLA, (SEQ ID NO: 1) which was described in De (1995) FEMS Micr. Let. 127:263-272. There is no description in the publication of using the expression plasmids in Pseudomonads, and the increase in expression of the hirudin using this strategy is only marginal when compared to control expression in E. coli. 
U.S. Pat. No. 5,641,671 to Unilever Patent Holdings B.V. is directed to the expression of a Pseudomonas lipase from P. glumae with a stabilizing protein and preferably a signal sequence endogenous to the lipase gene.
European Patent No. EP 0 121 352 to Atkinson et al. describes a cysteine free leader sequence from Pseudomonas species RS-16 carboxypeptidase to be used in a vector to enhance periplasmic expression of a protein. The patent discloses cysteine residues in the signal sequence can foster interaction with the cell membrane and retard secretion. The disclosed signal sequence is MRPSIHRTAIAAVLATAFVAGT (SEQ ID NO: 2).
Strategies that rely on signal sequences for targeting proteins out of the cytoplasm often produce improperly processed protein. This is particularly true for amino-terminal secretion signals such as those that lead to secretion through the Sec system. Proteins that are processed through this system often either retain a portion of the secretion signal, require a linking element which is often improperly cleaved, or are truncated at the terminus.
As is apparent from the above-described art, many strategies have been developed to target proteins to the periplasm of a host cell. However, known strategies have not resulted in consistently high yield of properly processed, active recombinant protein, which can be purified for therapeutic use. One major limitation in previous strategies has been the expression of proteins with poor secretion signal sequences in inadequate cell systems.
As a result, there is still a need in the art for improved large-scale expression systems capable of secreting and properly processing recombinant polypeptides to produce transgenic proteins in properly processed form. An ideal system produces high levels of soluble protein and allows targeting of that protein to the periplasm and potentially to the extracellular media.
An object of the invention is to provide a system and method of increasing expression of properly processed recombinant protein in a host cell.
Another object of the invention is to provide a system and method for increasing expression of active recombinant protein in a host cell.
Another object of the invention is to provide a commercial scale system with high levels of expression of recombinant, properly processed protein.